The overall objectives of this proposal are to elucidate the mechanism of action of different classes of next generation ?-secretase modulators (GSMs), to determine their synergistic effect, and to apply them in examining the role of A?42, A?38 and A?37 in amyloid pathology, synaptic plasticity and learning and memory. The development of GSMs that suppress ?-secretase activity for A?42 production and yet do not affect overall APP processing and cleavages of other substrates has emerged as a promising strategy for AD therapy. Progress in the development of these clinical candidates depends on a deeper understanding of the drug-target interactions. To this end we propose to map the binding site of acid GSMs within the ?-secretase complex and to investigate the structural basis of ?-secretase modulation. Additionally, we will determine the mechanism of cooperatively between the ?-secretase active site and the imidazole based GSM binding site(s). Finally, we will examine the synergistic effect of two classes of GSMs in cellular and animal models with a focus on safety, synaptic plasticity and learning and memory. The proposed research will provide mechanistic insights into GSM modulation of ?-secretase, the function of A? in disease and new therapeutic strategies, shaping our understanding of GSM selectivity and advancing our ability to design effective treatment.